Calcium-Binding Property of Dephosphorylated Caseins

Whole casein, α_s-casein and k-casein were dephosphorylated with a phosphoprotein phosphatase prepared from beef spleen and their calcium-binding capacities were compared with those of respective native caseins by a ultracentrifugal method.

The bindings of the calcium to 94% dephosphorylated whole casein and to 97 % dephosphorylated α_s-casein at neutral pH were approximately one third of those to respective native caseins. The decrease of calcium-binding capacity of k-casein due to dephosphorylation was also significant.

The effect of pH on the state and the calcium-binding capacity of dephosphorylated caseins was also examined and the role of organic phosphate groups of casein as calcium-binding sites was discussed.     

Antigenic Reactivity of Dephosphorylated αs1-Casein,Phosphopeptide from β-Casein and O-Phospho-l-serine towards the Antibody to Native αs1 -Casein

To study whether the phosphoserine residue is associated with the antigenicity of bovine α_s1- casein, we examined the antigenic reactivity of dephosphorylated α_s1-casein, peptide 1~25 from bovine β-casein and three chemical reagents with IgG antibody specific to native α_s1-casein by an enzyme-linked immunosorbent assay.

The reaction between native α_s1-casein and its IgG antibody was inhibited more strongly by native α_s1-casein than by dephosphorylated α_s1-casein. Peptide 1~25, having a phosphoserine residue-concentrated region from bovine β-casein, noticeably inhibited the reaction between native α_s1 -casein and its antibody. Furthermore, the O-phospho-l-serine residue inhibited the reaction of peptide 61~123 with anti-native α_s1-casein antibody, although l-serine and sodium phosphate showed no measurable inhibition.

These results suggest that the phosphoserine residue associated with part of an antigenic site in bovine α_sl-casein.     

Effect of 1-Anilino-8-naphthalenesulfonate on Properties and Associations of αs-, β- and κ-Caseins

It was indicated from fluorescence spectra and fluorescence titration that a hydrophobic probe, 1-anilino-8-naphthalenesulfonate (ANS), binds to casein components (α_s-, β- and κ-caseins). Fluorescence intensity and affinity of ANS-κ-casein complex were larger than that of ANS-α_s- and ANS-β-casein complexes. Enhancements of fluorescence intensity of complexes of casein components were observed by the addition of KCI or CaCl₂. Reason for the enhancement was postulated to be the increase of the quantum yield of the ANS fluorescence caused by the environmental change of ANS binding region of the casein components.

Marked increase of sedimentation coefficient of β-casein in the presence of KCl or CaCl₂ at 10°C was caused by the addition of ANS. This may be responsible for the stimulation of the Ca-dependent precipitation of β-casein by the addition of ANS.

It was found that α_s · κ-association was prevented by ANS and that hydrophobic interaction have an important role for α_s · κ-association.     

Characterization of a biosurfactant, mannosylerythritol lipid produced from Candida sp. SY16

One yeast strain, SY16, was selected as a potential producer of a biosurfactant, and identified as a Candida species. A biosurfactant produced from Candida sp. SY16 was purified and confirmed to be a glycolipid. This glycolipid-type biosurfactant lowered the surface tension of water to 29 dyne/cm at critical micelle concentration of 10 mg/l (1.5 × 10⁻⁵ M), and the minimum interfacial tension was 0.1 dyne/cm against kerosene. Thin-layer and high-pressure liquid chromatography studies demonstrated that the glycolipid contained mannosylerythritol as a hydrophilic moiety. The hydrophilic sugar moiety of the biosurfactant was determined to be β-d-mannopyranosyl-(1 → 4)-O-meso-erythritol by nuclear magnetic resonance (NMR) and fast atom bombardment mass–spectroscopy analyses. The hydrophobic moiety, fatty acids, of the biosurfactant was determined to be hexanoic, dodecanoic, tetradecanoic, and tetradecenoic acid by gas chromatography–mass spectroscopy. The structure of the native biosurfactant was determined to be 6-O-acetyl-2,3- di-O-alkanoyl-β-d-mannopyranosyl-(1 → 4)-O-meso-erythritol by NMR analyses. We newly determined that an acetyl group was linked to the C-6 position of the d-mannose unit in the hydrophilic sugar moiety. Received: 18 December 1999 / Received last revision: 2 June 1999 / Accepted: 4 June 1999     

The conformation and aggregation of bovine β-casein A. II. Thermodynamics of thermal association and the effects of changes in polar and apolar interactions on micellization

A sedimentation analysis has been used to determine the proportion of protein present as monomer and aggregate in 0.5 and 1.0 g/dl solutions of β-casein A in pH 7 phosphate buffer over the temperature range 10–40°C. The amount and molecular weight of the aggregate increase with temperature; under the conditions used, the aggregation number (n) of β-casein is given approximately by n = 0.6t + 2 with t in degrees centigrade. The concentration of β-casein in monomeric and aggregated states at different temperatures is used to calculate the standard enthalpy of aggregation ΔH° (Van't Hoff) by assuming that β-casein undergoes a cooperative, two-state, micellization process; aggregation is an endothermic process and ΔH° = 66.0 ± 2.6 kJ mol^?1. Combination of this ΔH° with the amount of protein calculated to dissociate when 1 g/dl solutions are diluted isothermally to 0.5 g/dl gives the heat of dilution at various temperatures. These calculated heats of dilution are compared with the experimental values obtained by carrying out the same dilutions in a microcalorimeter. The heat of dilution decreases linearly with β-casein concentration, but the extrapolated zero-concentration values of 65.8 ± 1.6 kJ mol^?1 is the same as the Van't Hoff enthalpy. This agreement in the enthalpy values indicates that the micellization of β-casein occurs cooperatively. The effect of modifying the hydrophobic/hydrophilic balance of the system on the micellization of β-casein A has been investigated. The hydrophobic interaction between the protein molecules is decreased by removing the three C-terminal residues (Ileu Ileu Val) with carboxypeptidase-A. This modification drastically reduces the ability of the β-casein molecule to form micelles. Substitution of ²H₂O for H₂O at constant temperature perturbs the monomer–micelle equilibrium in favor of micelles because of enhanced hydrophobic interactions in the former solvent. The results are consistent with β-casein micellization involving a delicate balance of the hydrophobic forces favoring aggregation and electrostatic forces opposing it.     

Amino Acid Sequence of Tremerogen A–10, a Peptidal Hormone,Inducing Conjugation Tube Formation in Tremella mesenterica Fr.

κ-Casein components having various carbohydrate contents were prepared by diethylaminoethyl-cellulose chromatography and the interactions of each κ-casein component both with α_s1-casein and with β-casein were examined by Sepharose 4B gel chromatography, ultra-centrifugal experiments and viscosity measurements. Each κ-casein component could form complex with α_s1- and β-casein in the absence and presence of CaCl₂. Molecular weight of complexes of unfractionated κ-casein both with α_s1-casein and with κ-casein were about 70 × 10⁴ at 37°C in the absence of CaCl₂, while those of complexes of each κ-casein component with α_s1 and β-casein were about 50 × 10⁴. Stokes radii of complexes increased with increasing calcium ion. While sedimentation coefficient at 37°C of complex with β-casein had almost the same value, those of complexes with α_s1-casein decreased with increase of carbohydrate content of κ-casein components. Intrinsic viscosity of complex of κ-casein component having much carbohydrate was almost the same among tested temperatures. It is suggested that heterogeneity of κ-casein is necessary to form large complex and that the carbohydrate moiety of κ-casein contributes the stability of casein complex.     

Determination of Acidic Component of Konjac Mannan

mRNA was isolated from mammary glands of lactating cow by affinity chromatography on poly(U)-Sepharose. The mRNA was heterogeneous on 3% agarose gel electrophoresis in the presence of 6m urea. The molecular weight of the main peak was estimated to be 3.3 x 10⁵. The mRNA was translated in a cell-free protein synthesizing system derived from wheat germ extract, and the translation products were analysed by the indirect immunoprecipitation method using specific antisera for casein components. About 50% of the total protein directed by this mRNA was casein. The relative amounts of α_s1-, β-,and k-casein in the translation products were nearly the same as those in bovine milk. The immunoprecipitates were analysed on sodium dodecyl sulfatepolyacrylamide gradient gel (15~20%) electrophoresis, and their mobilities were compared with those of dephosphorylated and non-glycosylated casein as standard, α_s1- and k Casein synthesized in vitro migrated more slowly than standard caseins, while synthesized β-casein migrated slightly faster than the standard β-casein.     

Role of Tyrosine Residues in Interactions between Casein Components

It was indicated from ultraviolet difference spectra and ultracentrifugal experiments that associations occurred between two casein components (α_s- and κ-caseins, β- and κ-caseins and α_s- and β-caseins) at lower CaCl₂ concentrations (2~3 mm) and that aromatic amino acid residues participated in the associations. Chemical modification studies with 2-hydroxy-5-nitrobenzylbromide indicated that tryptophane residues of each casein component were not essential for these associations. It was also demonstrated by nitration of tyrosine residues with tetranitromethane that tyrosine residues of κ-casein were essential for α_s·κ-association and for β·κ-association and that tyrosine residues of α_s-casein were important to α_s·β-association.

Interactions between casein components were also studied at higher CaCl₂ concentration (10 mm) which is enough for micelle formation. It was found that tyrosine residues of κ- casein played an important role for the stabilization of α_s- and β-caseins. Properties of the nitrated-β-casein were almost the same as that of the native β-casein except the absorption spectrum. α_s·β-Interaction in the presence of 10 mm CaCl₂ was investigated by use of the nitrated-β-casein instead of the native β-casein. It was proved that α_s-casein was stabilized by the nitrated-β-casein and that precipitation of the nitrated-β-casein increased in the presence of α_s-casein.

The mechanism of interactions between casein components at higher CaCl₂ concentration (10 mm) are discussed in connection with the associations at lower CaCl₂ concentrations (2~3 mm).     

Studies on Molecular Properties of αs1-κ-Casein Complex by the Hydrodynamical Methods

Some molecular properties of α_s1-κ-casein complex, α_s1- and κ-casein polymers were examined by gel filtration, ultracentrifugation, and viscometry at pH 7.1. The Stoke’s radii of α_s1-κ-casein complex, α_s1- and κ-casein polymers were 99, 44 and 108 Å, respectively. The molecular weight of the above proteins were approximately 45 × 10⁴, 10 × 10⁴ and 80 × 10⁴, respectively. The stokes radius of α_s1-κ casein complex reduced compared with that of κ-casein polymer and the molecular weight of the complex was about half that of κ-casein polymer. These results suggest that κ-casein polymer dissociates into 4 smaller particles when α_s1-κ-casein complex is formed. The frictional coefficient and Scheraga-Mandelkern constants for each protein suggest that the molecular shape of α_s1-casein polymer is globular and that of α_s1-κ-casein complex and κ-casein polymer is rod-like.     

Photolysis of Aryl Glycosides with Ultraviolet Irradiation

Bovine casein components (α_sl-, β-, and κ-caseins) were chemically phosphorylated and the properties of the modified components were compared with those of the native to clarify the function of the intrinsic phosphate groups of casein components in casein micelle formation. The calcium binding ability of casein components increased after chemical phosphorylation. The concentrations of calcium chloride required to precipitate modified α_sl- and β-caseins were higher than those for native components. However, phosphorylation of α_sl- and β-caseins did not affect their properties of forming micelles through interaction with κ-casein. The stabilizing ability of κ-casein for α_sl- and β caseins was impaired by its phosphorylation, but the stability was recovered by treating phosphorylated κ-casein with phosphoprotein phosphatase. The results show that the phosphate content of κ-casein must be low to form a stable casein micelle. The results also explain why the specific phosphorylation of casein components in the mammary gland is required.     

Post-translational Modification of κ-Casein in the Golgi Apparatus of Mid-lactating Mammary Glands from Rat and Cow

Bovine κ-casein, a phosphoglycoprotein, has mucin-type carbohydrate chains. Subcellular distribution of enzymes that take part in the post-translational modification of κ-casein was examined. In lactating mammary glands from rats and cows, N-acetyl-galactosaminyl transferase, galactosyl transferase, sialyl transferase, and casein kinase were localized specifically in the Golgi apparatus.

The substrate specificities indicate that these enzymes are actually responsible for the processing of κ-casein.

The presence of a phosphate group attached to κ-casein did not affect the rate of glycosylation by N-acetyl-galactosaminyl transferase, while the presence of carbohydrate chains attached to κ- casein strongly reduced the rate of phosphorylation by casein kinase. These results suggest that in the Golgi apparatus, phosphorylation of κ-casein precedes glycosylation.     

Studies on the Interaction between αs1- and β-Caseins

The interaction of α_s1-casein with β-, dephosphorylated β-,γ- and R-caseins was studied. It was proved by the sedimentation velocity experiments that α_s1-casein formed a complex with each of these components at 25±C in the presence of 3 mm CaCl₂.

In the presence of 10 mm CaCl₂, β- and dephosphorylated β-casein prevented the precipitation of α_s1-casein and gave micelle-like turbid solutions. However, γ- and R-caseins, fragments of β-casein, did not stabilize α_s1-casein. It was concluded from these results that α-casein interacted with α_s1-casein through its hydropholic region corresponding to R-casein and that hydrophilic region of β-casein was responsible for the stabilization of α_s1-casein.     

Post-translational phosphorylation affects the IgE binding capacity of caseins

IgE response specific to those molecular regions of casein that contain a major phosphorylation site was analyzed using native and modified caseins and derived peptides. This study included (i) the naturally occurring common variants A1 and A from beta- and alphas2-caseins, respectively, which were purified in the native form and then dephosphorylated, (ii) a purified rare variant D of alphas2-casein which lacks one major phosphorylation site, and (iii) the native and dephosphorylated tryptic fragment f(1-25) from beta-casein. Direct and indirect ELISA using sera from patients allergic to milk showed that the IgE response to caseins is affected by modifying or eliminating the major phosphorylation site.     

Properties of Acid Casein and Casein Micelles from Milk Destabilized by Frozen Storage

No difference was found in calcium sensitivity, electrophoretic and optical properties between acid caseins prepared from skimmilk before and after frozen storage (up to 180 days at ?7°C).

Destabilization of casein micelles can not be explained either by the reduction of solvation of the micelles or by the liberation of κ-casein from the micelles. However, when storage period was extended (about six months), splitting of a part of κ- and β-casein from the micelles to soluble form was observed, suggesting a drastic change of structure of the destabilized casein micelles.     

Studies on the Proteolytic Action of Dairy Lactic Acid Bacteria

In sterilized skim milk or sterilized 10% solution of dry skim milk at 120°C for 15 min, Lactobacillus bulgaricus, Lactobacillus helveticus and Streptococcus lactis were cultivated for 7 days at given temperature.

Both NCN (non casein type nitrogen) content and pH in each culture of lactic acid bacteria were rapidly decreased until 2 days after cultivation, But NCN content increased and the pH change got small after 3 days cultivation.

Caseins prepared from the cultures of these three kinds of lactic acid bacteria were examined electrophoretically. From the results of electrophoresis of these caseins, we have concluded that α-casein could be hydrolyzed by these lactic acid bacteria. And, it seemed that β-casein could not be hydrolyzed by these lactic acid bacteria.

Rennet easily hydrolyzed casein treated with L. bulgaricus and L. helveticus but hardly hydrolyzed that treated with S. lactis compared with control-casein. Caseins treated with L. bulgaricus and L. helveticus were hydrolyzed easier than control-casein.

Particle weights of caseins prepared from fermented milk by lactic acid bacteria, Streptococcus cremoris, Streptococcus lactis, Lactobacillus bulgaricus and Lactobacillus helveticus, and of hydrolyzed casein by rennet, trypsin or pepsin were measured according to the light scattering experiment.

Particle weights of various treated caseins were larger than that of raw native casein at both pH 7.0 and 12.0. And the heating caused the polymerization of casein to large particle.     

Removal of phosphate groups from casein with potato acid phosphatase.

Potato acid phosphatase (EC 3.1.3.2) was used to remove the eight phosphate groups from alphas1-casein. Unlike most acid phosphatases, which are active at pH 6.0 or below, potato acid phosphatase can catalyze the dephosphorylation of alphas1-casein at pH 7.0. Although phosphate inhibition is considerable (K1=0.42 mM phosphate), the phosphate ions produced by the dephosphorylation of casein can be removed by dialysis, allowing the reaction to go to completion. The dephosphorylated alphas1-casein is homogeneous on gel electrophoresis with a slower mobility than native alphas1-casein and has an amino acid composition which is identical to native alphas1-casein. Thus the removal of phosphate groups from casein does not alter its primary structure. Potato acid phosphatase also removed the phosphate groups from other phosphoproteins, such as beta-casein, riboflavin binding protein, pepsinogen, ovalbumin, and phosvitin.     

Conidial water affinity is an important characteristic for thermotolerance in entomopathogenic fungi

The interspecific thermotolerance of several species of entomopathogenic fungi was evaluated based on the conidial water affinity. The species were divided between hydrophilic and hydrophobic conidia. The species with hydrophobic conidia were Beauveria bassiana (ARSEF 252), Metarhizium brunneum (ARSEF 1187), Metarhizium robertsii (ARSEF 2575), Isaria fumosorosea (ARSEF 3889) and Metarhizium anisopliae s.l. (ARSEF 5749). The species with hydrophilic conidia were Tolypocladium cylindrosporum (ARSEF 3392), Tolypocladium inflatum (ARSEF 4877), Simplicillium lanosoniveum (ARSEF 6430), Lecanicillium aphanocladii (ARSEF 6433), S. lanosoniveum (ARSEF 6651), Aschersonia placenta (ARSEF 7637) and Aschersonia aleyrodis (ARSEF 10276). The conidial surface tension of each isolate was also studied. Conidial suspensions were exposed to 38, 41 or 45 °C. After exposure, the suspensions were inoculated on media and conidial germination was evaluated. Considerable differences in thermotolerance were found among the 12 entomopathogenic fungal species. Species with hydrophobic conidia were generally more thermotolerant than species with hydrophilic conidia. All isolates with hydrophobic conidia showed higher conidial surface tension than the isolates with hydrophilic conidia.     

Isolation of Tremerogen a-13, a Peptidal Sex Hormone of Tremella mesenterica

Bovine casein micelles were fractionated on controlled pore granule (CPG-10/3000) chromatography by size and the chemical properties of the fractionated micelles were compared. The results indicated the presence of two types of micelles distinguishable as large and small micelles. In skim milk, 72.7% of casein was calculated to be in the form of small micelles, 13.6% in the form of large micelles and 13.8% in non-micellar casein form.

The α_s1-casein content decreased, but β- and κ-casein content increased as the micelle size became smaller. κ-Casein in large micelles had a much higher sialic acid content than in small micelles. It was found that this difference in sialic acid content was due to the presence of non-glycosylated κ-casein in small micelles. In large micelles, non-glycosylated κ-casein was almost undetectable.

The addition of wheat germ lectin to micelles resulted in the formation of aggregates through intermicellar bridges between the carbohydrate chains of κ-casein located on the surface of the micelles. Both large and small micelles formed aggregates after the addition of wheat germ lectin. Large micelles were more sensitive to wheat germ lectin than small ones.     

Studies on the Proteolytic Action of Dairy Lactic Acicl Bacteria

Aseptic rennet curd prepared under the aseptic conditions and Str. cremoris- and L. helveticus-cheese prepared by sandwiching the cell pellets of Str. cremoris and L. helveticus between aseptic rennet curd, respectively, were ripened at 10°C for desired period.

Water soluble nitrogen (WSN) contents of both aseptic rennet curd and two kinds of cheese were determined. Gradual increase of WSN content of aseptic rennet curd was recognized all through the ripening preiod. WSN contents of both Str. cremoris- and L. helveticus-cheese were remarkably higher than those of aseptic rennet curd after 12 days ripening. This tendency was more remarkably recognized after 60 or 70 days ripening. α^s-Casein was mainly hydrolyzed by these lactic acid bacteria during ripening. α^s-Casein in two kinds of the cheese was more easily degradated by these lactic acid bacteria than that in aseptic rennet curd by rennet.

Judging from the results in previous and present reports, it was estimated that lactic acid bacteria used as a starter began to autolyze after 12 days ripening and that intracellular proteases released from their cells mainly hydrolyzed α^s-casein contained in Ca-paracaseinate of aseptic rennet curd to water soluble substances. This hydrolysis was also estimated from the viscous texture observed by scanning electron micrography.